Currently in the US alone there are 6 million units of platelets transfused each year, with the need for platelet transfusions appearing to increase with adoption of cell based therapy as a modality of treatment for cancer patients. For these patients, along with those experiencing bone marrow failures, platelet disorders or undergoing chemotherapy/radiotherapy, failure to provide platelet transfusion on a timely basis could be fatal. However current platelet transfusions rely solely on volunteer donors, limiting platelet collection. Such limitations can be theoretically overcome by having a master cell bank to produce an adequate number of platelets on demand. While proof of principle has been established for ex vivo generation of platelets from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), current platelet yields are insufficient for clinical applications. Furthermore, derivation of platelets and precursor megakaryocytes (MK) from hESC/iPSC is not without additional caveats, including ethical issues surrounding the use of ESC, concerns regarding the safety of iPSC, and the prolonged culture period required for hESC/iPSC to differentiate into MK progenitors/platelets (>3 weeks). We hypothesize that clinically relevant numbers of platelets can be derived from ex vivo expanded human cord blood (CB) cells in the presence of HDAC inhibitor (HDACi, i.e., VPA), avoiding the potential ethical, safety and practical concerns of using hESC/iPSCs. In order to pursue this hypothesis, we propose the following specific aims: 1) Optimize expansion of CB CD34+ cells while ensuring retention of MK differentiation potential. VPA as a prototype and additional HDACi will be tested along with other small molecules including StemRegenin 1 (SR1) and UM171 to further augment expansion of CD34+ cells retaining MK potential. 2) Determine epigenetic processes which augment proliferation and differentiation of MK from expanded CD34+ cells. Based on the epigenetic mechanism, which is identified by differential deacetylation assays comparing cells from CD34+ cell expansion displaying poor vs. optimal MK potential, cultures will be further optimized, for example through the use of isoform-selective HDACi synthesized in our collaborator's laboratory. 3) Optimize generation of MK/platelets from expanded CD34+ cells, and study the in vitro and in vivo function of the ex vivo generated MK/platelets. Successful execution of our proposed studies will allow for the optimization of our multistep strategy to produce MK/platelet from expanded CD34+ cells, leading to the generation of 3 to 4 units of platelets from a single CB unit. Mechanistic studies will further elucidate the underlying mechanisms of MK commitment and differentiation, potentially allowing for additional optimization of MK production. These studies may lead to a readily available source of MK progenitors or platelets generated ex vivo as a readily available source of platelets for transfusions, especially during national emergencies.